JP2024501829A - Crystal form III of melanocortin receptor agonist compound and method for producing the same - Google Patents

Abstract

The present invention relates to crystalline Form III of the compound represented by Formula 1, a method for producing the same, and a pharmaceutical composition containing the same. Crystalline Form III of the compound represented by Formula 1 of the present invention can be characterized by an XRPD pattern, TG/DTA profile, NMR spectrum and/or DVS profile.

Description

This application claims the benefit of priority based on Korean Patent Application No. 10-2020-0180809 dated December 22, 2020, and all contents disclosed in the documents of the Korean patent application are herein incorporated by reference. It will be incorporated as part of the book.

The present invention relates to crystal form III of a new compound that exhibits excellent melanocortin receptor enhancing activity, a method for producing the same, and a pharmaceutical composition containing the same.

Leptin protein is a hormone secreted by adipocytes, and the secretion amount increases with the increase in body fat content, and the secretion amount increases as the content of body fat increases. By regulating its function, it regulates various in-vivo functions, including appetite, body fat content, and energy metabolism (Schwartz, et al., Nature 404, 661-671 (2000)). Signal transduction of appetite and body weight regulation by the leptin protein is carried out through the regulation of various downstream factors, the most representative of which are melanocortin, AgRP (agoutirelated peptide), and neuropeptide Y ( neuropeptide Y (NPY) hormone.

When the concentration of leptin in the blood increases as a result of an overabundance of calories in the body, the secretion of the proopiomelanocortin (POMC) protein hormone in the pituitary gland increases, and the production of AgRP and NPY decreases. Decrease. A small peptide hormone, alpha-MSH (melanocyte stimulating hormone), is produced from POMC neurons, and this hormone acts as an agonist for Melanocortin-4 Receptor (MC4R) in secondary neurons, which stimulates appetite. resulting in a decrease. On the other hand, when the concentration of leptin decreases as a result of a calorie deficit, the expression of AgRP, an MC4R antagonist, increases, and the expression of NPY also increases, resulting in increased appetite. That is, alpha-MSH hormone and AgRP hormone are involved in appetite regulation by promoting and antagonistic roles for MC4R in response to changes in leptin.

Alpha-MSH hormone binds to three MCR subtypes in addition to MC4R and induces various physiological responses. To date, five types of MCR subtypes have been identified, among which MC1R is mainly expressed in skin cells and is involved in melanin pigment regulation (skinpigmentation), MC2R is mainly expressed in the adrenal gland, and MC2R is mainly expressed in the adrenal gland. It is known to be involved in the production of glucocorticoid hormone, and its only ligand is ACTH (adrenocorticotropic hormone) derived from POMC. MC3R and MC4R, which are mainly expressed in the central nervous system, are involved in regulating appetite, energy metabolism, and fat storage efficiency in the body, while MC5R, which is expressed in various tissues, is known to regulate exocrine function. (Wikberg, et al., Pharm Res 42(5) 393-420 (2000)). In particular, activation of the MC4R receptor has been proven to be the main point of action in the development of obesity treatment agents, as it shows the effect of efficiently reducing body weight by inducing a decrease in appetite and an increase in energy metabolism. (Review: Wikberg, Eur. J. Pharmacol 375, 295-310 (1999)); Wikberg, et al. , Pharm Res 42(5) 393-420 (2000); Douglas et al. , Eur J Pharm 450, 93-109 (2002); O'Rahilly et al. , Nature Med 10, 351-352 (2004)).

The role of MC4R in regulating appetite and body weight was first demonstrated by an animal model (agouti mouse) in which the agouti protein was abnormally expressed. In the case of agouti mice, the agouti protein is expressed at high concentrations in the central nervous system due to genetic mutations, and plays the role of an antagonist of MC4R in the hypothalamus, thereby inducing obesity. (Yen, TT et al., FASEB J. 8, 479-488 (1994); Lu D., et al. Nature 371, 799-802 (1994)). Subsequent research results have shown that AgRP (agouti-related peptide), which is similar to agouti protein, is actually expressed in hypothalamic nerves, and these are also known to be involved in appetite regulation as antagonists to MC4R. (Shutter, et al., Genes Dev., 11, 593-602 (1997); Ollman, et al. Science 278, 135-138 (1997)).

Cerebral administration of alpha-MSH, an in vivo MC4R agonist, to animals has been shown to reduce appetite, whereas MC4R antagonists SHU9119 (peptide) or HS014 (peptide) A phenomenon has been observed in which the appetite is also increased when treated with . In addition, in animal studies using Melanotan II (MTII, Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH2) and its similar agonist HP228, cerebral, intraperitoneal or subcutaneous After administration, the efficacy of appetite suppression, weight loss, and increase in energy metabolism has been confirmed (Thiele TE, et al. Am J Physiol 274 (1 Pt 2), R248-54 (1998); Lee M.D., et al. FASEB J 12, A552 (1998); Murphy B., et al. J Appl Physiol 89, 273-82 (2000)). In contrast, administration of representative SHU9119 to animals showed significant and sustained food intake and weight gain, providing pharmacological evidence that MCR agonists can be anti-obesity agents. Although the anorexic effect that appears markedly when MTII is administered does not appear in MC4R KO (knock-out) mice, this experimental result also proves that the anorexic effect is mainly caused by activation of MC4R. (Marsh, et al., Nat Genet 21, 119-122 (1999)).

The main types of obesity treatment drugs that have been developed to date have been appetite suppressants that act on the central nervous system, and most of these have been drugs that modulate the actions of neurotransmitters. Examples include the noradrenalin agents phentermine and mazindol, and the serotonergic agents fluoxetine and sibutramine. However, in the case of the nerve signal transmitter modulating agent, it exerts a wide range of effects on various physiological effects in addition to appetite inhibition due to its many subtypes of receptors. Therefore, the regulator lacks selectivity for each subtype, and when administered for a long period of time, it causes various side effects, which is a major drawback.

On the other hand, melanocortin agonists are not neurosignal transmitters but neuropeptides, and considering that all other functions other than energy metabolism are normal in MC4R gene KO mice, they do not affect other physiological functions. , has an advantage as a point of action in that it can induce only weight loss due to appetite suppression. In particular, as the receptor is a G-protein coupled receptor (GPCR), which belongs to the most successful category of new drug action points developed to date, it is necessary to ensure selectivity for subtype receptors. It is largely distinguished from existing points of action in that it is relatively easy to use.

As examples of utilizing such melanocortin receptors as sites of action, International Publication Nos. WO2008/007930 and WO2010/056022 disclose compounds as agonists of melanocortin receptors.

In addition, the inventors of the present invention have conducted intensive research and have discovered a novel compound represented by the following chemical formula 1 that has excellent selective enhancing activity for melanocortin receptors, particularly melanocortin-4 receptor (MC4R), and a method for producing the same. (Korean Application No. 10-2019-0141649 (filed on November 7, 2019)).

(R ₁ is C ₂ -C ₅ alkyl.)

On the other hand, the crystal structure of a pharmaceutically active ingredient sometimes influences the chemical stability of the drug. Different crystallization and storage conditions can change the crystal structure of the compound, sometimes resulting in production with different forms of crystalline forms. In general, amorphous drug products do not have a regular crystal structure and sometimes have other characteristics such as poor product stability, small particle size, difficult filtration, susceptibility to agglomeration and poor flow properties. It has some defects. Therefore, there is a need to improve various physical properties of the product. Thus, for a single compound, there is a need to study crystal structures with high purity and good chemical stability.

International patent application publication number WO2008/007930 International patent application publication number WO2010/056022

An object of the present invention is to provide a stable crystalline form of a novel compound that has excellent selective enhancing activity against melanocortin receptors, particularly melanocortin-4 receptor (MC4R), and a method for producing the same.

Another object of the invention is to provide pharmaceutical compositions containing stable crystalline forms of said novel compounds.

In order to achieve the above purpose,
In one aspect, the present invention provides crystalline Form III of the compound of Formula 1 below, a pharmaceutically acceptable salt thereof, or a solvate thereof, which has an X-ray powder diffraction (XRPD) pattern. , the following diffraction angles (2θ values): 6.238±0.2°, 8.257±0.2°, 8.828±0.2°, 14.637±0.2°, 16.618±0 .2°, 17.465±0.2°, 18.859±0.2°, 19.061±0.2°, 19.333±0.2°, 20.642±0.2°, 22 Crystalline Form III is provided having three or more characteristic peaks selected from .679±0.2° and 25.985±0.2°.

In the chemical formula 1,
R ₁ is C ₂ -C ₅ alkyl.

The compound of Formula 1 may have an asymmetric carbon center, an asymmetric axis, or an asymmetric plane, and may have a cis or trans isomer, an R or S isomer, a racemate, a mixture of partial stereoisomers, and an individual partial stereoisomer. It can exist as isomers, and all these isomers and mixtures are included within the scope of the compound of Formula 1 above.

For convenience, unless otherwise specified, the compound of Formula 1 is used herein to include all compounds of Formula 1, pharmaceutically acceptable salts thereof, isomers thereof, and solvates thereof.

In one embodiment according to the present invention, R ₁ in Formula 1 is C ₂ -C ₅ alkyl. In another embodiment according to the present invention, R ₁ in Formula 1 is a linear or branched C ₂ -C ₅ alkyl, such as ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl or tert-butyl.

In another embodiment according to the present invention, R ₁ in Formula 1 is C ₂ -C ₄ alkyl. In another embodiment according to the present invention, R ₁ in Formula 1 is a linear or branched C ₂ -C ₄ alkyl, such as ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl and tert-butyl. Specifically, R ₁ may be iso-propyl.

In one embodiment according to the invention, the pharmaceutically acceptable salts are inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, tartaric acid, formic acid, citric acid, Organic carboxylic acids such as acetic acid, dichloroacetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid or naphthalenesulfonic acid These include, but are not limited to, acid addition salts formed by, for example,

In one embodiment according to the invention, the solvate is a hydrate; methanol, ethanol, 2-propanol, 1,2-propanediol, 1,3-propanediol, n-butanol, 1,4 -Butanediol, tert-butanol, acetic acid, acetone, butyl acetate, methyl acetate, ethyl acetate, propyl acetate, t-butyl acetate, isobutyl acetate, methyl ethyl ketone, 2-pentanone, tetrahydrofuran, acetonitrile, formamide, N,N-dimethylformamide , N-methyl-2-pyrrolidone, chloroform, toluene and mixtures thereof.

In one embodiment of the present invention, the crystalline form III may be a crystalline form of a solvate of the amide solvent represented by Formula 1.

In another embodiment of the present invention, the crystalline form III may be a crystalline form of the formamide solvate of Formula 1.

In one embodiment according to the invention, crystalline form III has an X-ray powder diffraction pattern of 6.238±0.2°, 8.257±0.2°, 8.828±0.2°, 14.637° ±0.2°, 16.618±0.2°, 17.465±0.2°, 18.859±0.2°, 19.061±0.2°, 19.333±0.2° , 20.642±0.2°, 22.679±0.2° and 25.985±0.2°, 3 or more, 5 or more, 7 or more, 9 or more, 10 or more, or It can have 11 or more characteristic peaks.

In one embodiment according to the invention, crystalline form III has an X-ray powder diffraction pattern of 6.238±0.2°, 8.257±0.2°, 8.828±0.2°, 14.637° ±0.2°, 16.618±0.2°, 17.465±0.2°, 18.859±0.2°, 19.061±0.2°, 19.333±0.2° , 20.642±0.2°, 22.679±0.2° and 25.985±0.2°.

Crystalline Form III according to the present invention may be a crystalline form of a solvate of the compound of Formula 1, and thus a halo due to residual solvent may be observed during XRPD measurements. For example, an XRPD peak observed at a 2θ angle of 20-30° can be a halo due to residual solvent, eg, formamide.

In one embodiment according to the invention, the crystalline Form III may have an X-ray powder diffraction (XRPD) pattern as shown in FIG.

Crystal form III according to the invention can exhibit two endothermic peaks at 80-130°C and 200-260°C in the differential scanning calorimetry (DSC) profile.

In particular, crystalline Form III according to the invention may contain an endothermic peak (200-260° C.) due to the solvent, for example formamide, in the differential scanning calorimetry (DSC) profile.

Further, crystal form III according to the present invention has a DSC profile of 10% or less, for example, 5% or less, 4% or less, 3% or less, 2% or less, 1 It can have a weight loss of less than .5% or 1.2%.

Crystal form III according to the invention also has a weight loss of 20-40%, such as 25%-30%, or 27.9%, in the range where the DSC profile shows an endothermic peak at 200-260°C. be able to.

In one embodiment according to the invention, the crystalline Form III can have the TG/DTA propyl shown in FIG.

Crystal form III according to the present invention may contain a peak due to a solvent, such as formamide, in the NMR analysis result.

In one embodiment according to the invention, said crystalline Form III may have the NMR results shown in FIG.

In this specification,
X-ray powder diffraction (XRPD) analysis is shown using a PANalytical X' Pert Pro MPD system, Malvern Panalytical Ltd.

Thermogravimetric analysis (TG/DTA) is performed using TGA/DSC1, Mettler-Toledo AG.

Nuclear magnetic resonance (NMR) results are shown using a Bruker 500MHz.

The crystalline form III may have higher purity and be more physically and chemically stable than the crude compound of formula 1, the amorphous compound of formula 1, or other crystalline forms of the compound of formula 1. It can be done.

In addition, crystalline Form III of the compound of Formula 1 has a higher ability to enhance melanocortin-4 receptors and to prevent diseases such as obesity, diabetes, inflammation, and erectile dysfunction than known melanocortin-4 receptor agonists. Alternatively, the therapeutic effect may be even better, but the effects of the present invention are not limited thereto.

In another aspect, the present invention provides a method for producing crystalline form III, comprising the steps of dissolving the compound of formula 1 in a crystallization solvent to produce a mixed solution, and obtaining crystals from the mixed solution. provide.

First, the compound represented by Formula 1 is dissolved in a crystallization solvent.

The compound of Formula 1 for preparing the crystal form III may be a compound of Formula 1, a salt thereof, an isomer thereof, or a solvate thereof.

The compound of Formula 1 can be obtained by the manufacturing method described in the specification of Korean Application No. 10-2019-0141649 (filed on November 7, 2019).

The crystallization solvent can be used without any particular restriction as long as it is a suitable solvent for crystallizing the compound. In one embodiment, the crystallization solvent includes a polar organic solvent.

The polar organic solvent may include, but is not limited to, an amide solvent.

The amide solvent may include, but is not limited to, formamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone, or a mixture thereof.

In one embodiment of the present invention, the polar organic solvent may include formamide.

In one embodiment according to the invention, the step of obtaining the crystals may be performed by precipitation with anti-solvent.

In the anti-solvent crystallization method, the crystallization solvent for dissolving the compound of Formula 1 may be the above-mentioned polar organic solvent, such as formamide, and the anti-solvent may be a non-polar organic solvent. At least one of an organic solvent and water can be used. The non-polar organic solvent can be used without any particular restriction as long as it has non-polar characteristics; for example, hexane, heptane, cyclohexane, carbon tetrachloride, benzene, chloroform, etc. can be used. .

In one embodiment according to the present invention, crystalline form III can be obtained by dissolving the compound of formula 1 in formamide and then adding heptane as an anti-solvent dropwise.

In another embodiment according to the invention, the step of obtaining the crystals may be performed by a slurry experiment.

The slurry method involves dissolving the compound of Formula 1 in a sufficient amount of solvent at a desired temperature until undissolved solid remains, sealing the compound, and stirring for a predetermined period while maintaining the temperature. However, it can be carried out by a method of filtration and drying, but is not limited thereto.

In the slurry method, the crystallization solvent for dissolving the compound of Formula 1 may be the polar organic solvent described above, such as formamide, but is not limited thereto.

In one embodiment according to the present invention, the compound of formula 1 is added to a sufficient amount of crystallization solvent at a constant temperature of 0° C. to 80° C. until solid remains, and then the compound is sealed and the temperature is increased. By maintaining and stirring for 1 to 60 days, crystalline form III can be obtained.

In the slurry method, the certain temperature is, for example, 0°C to 80°C, 5°C to 60°C, 5°C to 50°C, 5°C to 20°C, 20°C to It can be 50°C, 15°C to 60°C, 5°C, 20°C or 50°C.

In the slurry method, the period during which the compound dissolved in the crystallization solvent is maintained is not limited to, for example, 1 to 60 days, 1 to 30 days, 1 to 15 days, It can be from 1 to 10 days, from 5 to 7 days, or from 6 days.

The crystalline Form III obtained as described above can be of higher purity than the crude compound of Formula 1, the amorphous compound of Formula 1 or any crystalline form of Formula 1, and is physically and chemically Although it may be more stable, the effects of the present invention are not limited thereto.

In yet another aspect, the invention provides a pharmaceutical composition comprising (i) crystalline Form III as described above; and (ii) a pharmaceutically acceptable carrier.

Crystalline Form III according to the present invention exhibits an excellent enhancing effect on melanocortin receptors, particularly melanocortin-4 receptor (MC4R), and the present invention includes the above-mentioned Crystalline Form III as an active ingredient. A pharmaceutical composition for enhancing melanocortin receptor function can be provided. Specifically, the pharmaceutical composition can be a composition for enhancing melanocortin-4 receptor function.

In addition, the pharmaceutical composition can exhibit excellent effects on the prevention or treatment of obesity, diabetes, inflammation, and erectile dysfunction, and can be used for the prevention or treatment of obesity, for the prevention or treatment of diabetes, for the prevention or treatment of inflammation. The composition can be used for treatment or prevention or treatment of erectile dysfunction, but the use of the present invention is not limited to these diseases.

As used herein, "carrier" refers to a compound that facilitates introduction of a compound into a cell or tissue.

When administering Crystalline Form III of the present invention for clinical purposes, the total daily dose administered to the host in single or separate doses is preferably in the range of 0.01 to 10 mg/kg body weight; The specific dose level for a patient will vary depending on the specific compound used, the patient's weight, sex, health status, diet, time of drug administration, method of administration, excretion rate, drug mix, and disease severity. can do.

Crystal form III of the present invention can be administered by any route depending on the purpose. For example, amorphous compounds of the invention can be administered by injection or orally.

The pharmaceutical compositions of the present invention can be in various oral administration forms such as tablets, pills, powders, capsules, granules, syrups or emulsions, or for intramuscular, intravenous or subcutaneous administration. It can be in the form of parenteral administration, such as an injectable preparation.

Injectable formulations can be prepared according to known techniques using suitable dispersing agents, wetting agents, suspending agents, or excipients.

Excipients that can be used in the pharmaceutical formulation of the present invention include sweeteners, binders, solubilizers, solubilizers, wetting agents, emulsifiers, tonicity agents, adsorbents, disintegrants, antioxidants, and preservatives. These may include, but are not limited to, agents, lubricants, fillers, fragrances, and the like. For example, excipients include lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, silica, magnesium aluminum silicate, starch, gelatin, gum tragacanth, alginic acid, sodium alginate, methylcellulose, sodium carboxymethylcellulose, water. , ethanol, polyethylene glycol, polyvinylpyrrolidone, sodium chloride, calcium chloride, orange essence, strawberry essence, vanilla scent, etc. can be used.

When the pharmaceutical composition of the invention is in the form of oral administration, examples of carriers used include cellulose, calcium silicate, corn starch, lactose, sucrose, dextrose, calcium phosphate, stearic acid, magnesium stearate, stearic acid, Examples include, but are not limited to, calcium phosphate, gelatin, and talc.

When the pharmaceutical composition of the present invention is in the form of an injectable preparation, examples of the carrier include water, saline, aqueous glucose solution, aqueous pseudosugar solution, alcohol, glycol, ether, oil, fatty acid, fatty acid ester, glyceride, and the like. These include, but are not limited to:

In yet another aspect, there is provided crystalline Form III as described above for use in melanocortin receptor, particularly melanocortin-4 receptor (MC4R) hyperactivity applications.

In one embodiment, there is provided crystalline Form III as described above for use in the treatment or prevention of obesity, diabetes, inflammation or erectile dysfunction.

In yet another aspect, there is provided a method for enhancing the function of melanocortin receptors, particularly melanocortin-4 receptor (MC4R), which comprises the step of administering the above crystalline Form III to a subject.

In yet another aspect, a method of treating obesity, diabetes, inflammation, or erectile dysfunction is provided comprising administering to a subject Crystalline Form III as described above.

Since crystal form III according to the present invention exhibits an excellent enhancing effect on melanocortin receptors, particularly melanocortin-4 receptor (MC4R), it is useful for the prevention or treatment of obesity, diabetes, inflammation, and erectile dysfunction. can be used.

Crystal form III according to the present invention exhibits an on-target effect on melanocortin-4 receptors, thereby exhibiting a weight-reducing and food-reducing effect, and having no effect on anxiety and depression. It can be administered without safety problems such as side effects or induction of mutations caused by inhibition of the related gene.

Crystalline Form III according to the present invention also has superior purity, yield, physical and chemical stability compared to the crude compound of Formula 1, the amorphous compound of Formula 1 or any other crystalline form of Formula 1. Excellent.

Specifically, the crystalline form III may have superior solubility, storage stability, and production stability compared to the compound of chemical formula 1, the amorphous compound of chemical formula 1, or any other crystalline form of chemical formula 1. can.

3 is an XRPD result graph of Example 1. 3 is a TG/DTA result graph of Example 1. 1 is an NMR result graph of Example 1.

Hereinafter, the present invention will be explained in more detail with reference to production examples and examples. However, these Examples are illustrative of the present invention, and the scope of the present invention is not limited thereto.

Production Example 1: Production of methyl (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylate hydrochloride

The title compound was obtained through the following steps A, B, C, D and E.

Step A: Preparation of 1-(tert-butyl)2-methyl(2S,4S)-4-azidopyrrolidine-1,2-dicarboxylate Under nitrogen, 1-(tert-butyl)2-methyl(2S) ,4R)-4-((methylsulfonyl)oxy)pyrrolidine-1,2-dicarboxylate (48.5 g, 150 mmol) was dissolved in N,N'-dimethylformamide (250 ml), and sodium azide (19. 5g, 300ml) was added. After stirring at 80° C. for 16 hours and concentrating the reaction solvent under reduced pressure, water was added and extraction was performed twice with ethyl acetate. The organic layer was washed with an aqueous sodium chloride solution and water, and then dried and filtered over anhydrous magnesium sulfate. The filtrate was concentrated under reduced pressure to obtain crude 1-(tert-butyl)2-methyl(2S,4S)-4-azidopyrrolidine-1,2-dicarboxylate (39.59 g, 98%). and used in the next step without purification.

MS [M+H] = 271 (M+1)
¹ H NMR (400 MHz, CD3OD) δ 4.43-4.37 (m, 1H), 4.35-4.27 (br, 1H), 3.77 (s, 1.8H), 3.76 (s, 1.2H), 3.73-3.66 (m, 1H), 3.44-3.38 (m, 1H), 2.63-2.49 (m, 1H), 2.19-2.11 (m, 1H), 1.50 (s, 4.5H), 1.44 (s, 4.5H)

Step B: Preparation of 1-(tert-butyl)2-methyl (2S,4S)-4-aminopyrrolidine-1,2-dicarboxylate 1-(tert-butyl)2-methyl obtained in step A above After dissolving (2S,4S)-4-azidopyrrolidine-1,2-dicarboxylate (24.59 g, 91.0 mmol) in tetrahydrofuran (180 ml), 1M trimethylphosphine tetrahydro solution (109.2 ml, 109.2 mmol) was gradually added. After stirring at the same temperature for 1 hour, stirring was continued at room temperature for 3 hours. After the reaction solvent was concentrated under reduced pressure, dichloromethane (100 ml) and water (150 ml) were added and stirred for about 30 minutes. After separating the layers and extracting once again with dichloromethane, the organic layer was dry filtered over anhydrous magnesium sulfate. The filtrate was concentrated under reduced pressure to obtain crude 1-(tert-butyl)2-methyl(2S,4S)-4-aminopyrrolidine-1,2-dicarboxylate (20.62 g, 93%), which was purified. used in the next step without.

MS [M+H] = 245 (M+1)
^1H NMR (400 MHz, CD3OD) δ 4.27 (m, 1H), 3.77 (s, 1.8H), 3.76 (s,1.2H), 3.75-3.67 (m, 1H), 3.50-3.42 (m, 1H), 3.22-3.17 (m, 1H), 2.58-2.47 (m,1H), 1.82-1.71 (m, 1H), 1.48 (s, 4.5H), 1.42 (s, 4.5H)

Step C: Preparation of 1-(tert-butyl)2-methyl(2S,4S)-4-(((1s,4R)-4-methylcyclohexyl)amino)pyrrolidine-1,2-dicarboxylate Above Step B 1-(tert-butyl)2-methyl(2S,4S)-4-aminopyrrolidine-1,2-dicarboxylate (20.62 g, 84.4 mmol) obtained in was dissolved in dichloroethane (150 ml), 4-Methylcyclohexanone (9.5ml, 101.3mmol) was added. Sodium triacetoxyborohydride (26.8 g, 126.6 mmol) was added at 0°C, and the mixture was stirred at room temperature for 16 hours. The reaction solvent was concentrated under reduced pressure, water was added, and the mixture was extracted twice with ethyl acetate. The organic layer was washed with an aqueous sodium chloride solution and then dried and filtered over anhydrous magnesium sulfate. The filtrate was concentrated under reduced pressure and purified by column chromatography to obtain 1-(tert-butyl)2-methyl(2S,4S)-4-(((1s,4R)-4-methylcyclohexyl)amino)pyrrolidine-1. ,2-dicarboxylate (22.9 g, 80%) was obtained.

MS [M+H] = 341 (M+1)
^1H NMR (400 MHz, CD3OD) δ 4.26 (m, 1H), 3.76 (s, 1.8H), 3.75 (s, 1.2H), 3.78-3.71 (m, 1H), 3.49-3.40 (m, 1H), 3.22-3.16 (m, 1H), 2.69-2.60(br, 1H), 2.58-2.46 (m, 1H), 1.87-1.77 (m, 1H), 1.73-1.63 (m, 1H), 1.62-1.35(m , 8H), 1.48 (s, 4.5H), 1.42 (s, 4.5H), 0.96 (d, 3H)

Step D: Preparation of 1-(tert-butyl)2-methyl(2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-1,2-dicarboxylate 1-(tert-butyl)2-methyl(2S,4S)-4-(((1s,4R)-4-methylcyclohexyl)amino)pyrrolidine-1,2-dicarboxylate (obtained in step C) After dissolving 37.29 g, 109.5 mmol) in dichloromethane (500 ml) and adding triethylamine (61.1 ml, 438.1 mmol), isobutyryl chloride (11.7 ml, 219 mmol) was gradually added at 0°C. did. After stirring at room temperature for 16 hours, the reaction solvent was concentrated under reduced pressure, an aqueous sodium bicarbonate solution was added, and the mixture was extracted twice with ethyl acetate. The organic layer was washed with an aqueous sodium chloride solution and water, and then dried and filtered over anhydrous magnesium sulfate. The filtrate was concentrated under reduced pressure and purified by column chromatography to obtain 1-(tert-butyl)2-methyl(2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramide). Pyrrolidine-1,2-dicarboxylate (38.79 g, 86%) was obtained.

MS [M+H] = 411 (M+1)
^1H NMR (400 MHz, CD3OD) δ 4.27 (m, 1H), 3.76 (s, 1.8H), 3.75 (s, 1.2H), 3.78-3.72 (m, 1H), 3.50-3.41 (m, 1H), 3.33-3.14 (m, 1H), 2.69-2.60 (m, 2H), 2.57-2.43 (m, 1H), 1.87-1.79 (m, 1H), 1.70-1.61 (m, 1H), 1.60-1.32 (m , 8H), 1.47 (s, 4.5H), 1.41 (s, 4.5H), 1.10 (dd, 6H), 0.99 (d, 3H)

Step E: Preparation of methyl (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylate hydrochloride 1-(obtained in step D above) tert-butyl)2-methyl(2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-1,2-dicarboxylate (34.0 g, 82.8 mmol ) was dissolved in dichloromethane (200 ml), and then a 4N hydrochloric acid solution in 1,4-dioxane (82.8 ml, 331.3 mmol) was added at 0°C. After stirring at room temperature for 6 hours, the reaction solvent was concentrated under reduced pressure to obtain crude (28.7 g, 99%), which was used in the next step without purification.

MS[M+H] = 311 (M+1)

Production Example 2: Production of (3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carboxylic acid

The title compound was obtained by the method described in International Publication No. WO2004/092126.

MS[ M+H] = 282 (M+1)
^1H NMR (400 MHz, CD3OD) δ 7.43-7.33 (m, 4H), 3.90-3.69 (m, 3H), 3.59 (dd, J = 11.2, 10.0 Hz, 1H), 3.29 (dd, J = 11.2, 11.2 Hz, 1H), 3.18-3.09 (m, 1H), 1.44 (s, 9H)

Production Example 3: N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4 -Carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide production (MC70)

The title compound was obtained through the following steps A, B and C.

Step A: Methyl(2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-((1s, Production of 4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylate Methyl (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl) obtained in Production Example 1 isobutyramido)pyrrolidine-2-carboxylate hydrochloride (28.7g, 82.73mmol), (3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine obtained in Production Example 2 -3-carboxylic acid (24.5 g, 86.87 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (22.2 g, 115.83 mmol) and 1-hydroxybenzotriazole hydrate (15 .7 g, 115.83 mmol) was dissolved in N,N'-dimethylformamide (400 ml) and N,N'-diisopropylethylamine (72.0 ml, 413.66 mmol) was slowly added. After stirring at room temperature for 16 hours and concentrating the reaction solvent under reduced pressure, a 0.5N aqueous sodium hydroxide solution was added and extracted twice with ethyl acetate. The organic layer was washed twice with an aqueous sodium chloride solution and twice with water, and then dried and filtered over anhydrous magnesium sulfate. The filtrate was concentrated under reduced pressure and purified by column chromatography to give methyl (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl. )-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylate (41.19 g, 87%) was obtained.

MS [M+H] = 575 (M+1)

Step B: (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-((1s,4R) )-4-Methylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylic acid Production of methyl(2S,4S)-1-((3S,4R)-1-(tert-butyl)-4 obtained in step A above) -(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramide)pyrrolidine-2-carboxylate (39.4 g, 68.62 mmol) in methanol (450 ml), 6N aqueous sodium hydroxide solution (57.2 ml, 343.09 mmol) was added. After stirring at room temperature for 16 hours and adjusting the pH to about 5 with a 6N aqueous hydrochloric acid solution, the reaction solution was concentrated under reduced pressure. After dissolving the concentrate with dichloromethane, undissolved solids were filtered through a paper filter. The filtrate was concentrated under reduced pressure to obtain the crude title compound (38.4 g, 99%), which was used in the next step without purification.

MS [M+H] = 561 (M+1)

Step C: N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4- Preparation of (carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide (2S,4S)-1-((3S,4R)-1 obtained in step B above -(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramide)pyrrolidine-2-carboxylic acid (38. 4g, 68.60mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (18.4g, 96.04mmol) and 1-hydroxybenzotriazole hydrate (13.0g, 96.04mmol). After dissolving in N,N'-dimethylformamide (200 ml), morpholine (5.9 ml, 68.80 mmol) and N,N'-diisopropylethylamine (59.7 ml, 343.02 mmol) were gradually added in this order. After stirring at room temperature for 16 hours and concentrating the reaction solution under reduced pressure, 0.5N aqueous sodium hydroxide solution was added and extracted twice with ethyl acetate. The organic layer was washed twice with an aqueous sodium chloride solution and twice with water, and then dried and filtered over anhydrous magnesium sulfate. The filtrate was concentrated under reduced pressure and purified by column chromatography to obtain N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine- 3-Carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide (37.05g, 86%, MC70) was obtained. .

MS [M+H] = 630 (M+1)

[Example] N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4 Preparation of crystalline form III of -carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide

Example 1: Production by anti-solvent method After dissolving 25 mg of the compound (MC70) produced in Production Example 3 in formamide, 19 times the volume of formamide was added at room temperature using heptane as an anti-solvent. Thereafter, it was stored at 4° C. for 7 to 14 days, filtered, and dried to obtain the title compound (MC70 crystal form III).

Example 2: Production by Slurry Method 25 mg of the compound (MC70) produced in Production Example 3 above was added to a sufficient amount of formamide at the target temperature (5°C) until undissolved solids remained. After the test tube was sealed, the target temperature (5° C.) was maintained and stirred for 6 days. Thereafter, filtration and drying were performed to obtain the title compound (MC70 crystal form III).

Example 3: Production by slurry method The title compound (MC70 crystal form III) was obtained in the same manner as in Example 2, except that the target temperature was set at 20°C.

Example 4: Production by slurry method The title compound (MC70 crystal form III) was obtained in the same manner as in Example 2, except that the target temperature was set at 50°C.

The compounds obtained in Examples 1 to 4 were subjected to XRPD, TG/DTA, NMR, and DVS analysis using the following methods, and the analysis results showed that the compounds obtained in Examples 1 to 4 The compounds were confirmed to have the same crystalline form.

Graphs of XRPD (Figure 1), TG/DTA (Figure 2) and NMR (Figure 3) analyzes conducted on Example 1 are shown in Figures 1 to 4 to represent the compounds of Examples 1 to 4. Illustrated in FIG. In addition, XRPD analysis was also performed after the DVS measurement, and the results, which confirmed that the crystal form did not change, are illustrated in FIG.

Experimental example 1. XRPD Evaluation A powder XRPD diffraction pattern was obtained in the following manner using a PANalytical X'Pert Pro MPD system equipped with a monochromatized radiation source and a Ni filter as a solid phase detector.

After placing approximately 20 to 30 mg of the sample uniformly on a glass sample holder so that it has a flat surface, the generator of the instrument was set to 45 kV (acceleration voltage) and 40 mA (filament emission), and then , reflection mode (not-spin). The Bragg angle (2θ) was measured in the range of 4-40° with a step size of 0.026° and a time per step of 51 seconds.

The results of measuring the obtained crystal form III by XRPD are illustrated in FIG.

As confirmed from the spectrum shown in FIG. 1, crystal form III according to the present invention was confirmed to be a crystalline substance, and specific values of XRPD are shown in Table 1 below. The amorphous pattern observed at 20-30° 2θ was interpreted to be due to remaining unsolvated formamide.

Experimental example 2. Thermogravimetric analysis (TG/DTA)
TG/DTA was measured using a Mettler Toledo DSC1 system. Approximately 2 to 5 mg of the sample was weighed, placed in 40 μL Al crucible (flat-bottomed aluminum pan with one pin-hole lid), weighed accurately, and placed into the TG furnace. The sample was then heated at a rate of 10°C/min up to 300°C and stabilized at 30°C for 1 minute before heating. During measurements, nitrogen gas is supplied into the equipment at a rate of 70 mL/min to prevent oxygen and other gases from entering. Data collection and evaluation were performed using the software STARe.

The TG/DTA measurement results of the obtained crystal form III are illustrated in FIG.

As confirmed from FIG. 2, crystal form III contains 1.2% w/w (0.17 mol, eq. formamide) and 27.9% w/w (5.4 mol. eq. formamide), respectively. Two endothermic peaks were observed at about 95.08°C (Onset) and about 216.53°C (Onset) along with weight loss. Here, the temperature value may have an error of ±5°C.

The temperature at which the endothermic peak at about 216.53°C (Onset) is shown coincides with the boiling point of formamide, and formamide is also confirmed in the NMR results below, indicating that the crystal form III is a solvated formamide. I confirmed that it was a thing.

Experimental example 3.1H nuclear magnetic resonance spectrum (NMR)
Using the obtained crystal form III, NMR analysis was performed on a Bruker at 500 MHz using methanol d4 solvent, and the results are illustrated in FIG.

As a result of NMR analysis, crystal form III was confirmed to be a formamide solvate of Chemical Formula 1.

Claims (15)

Crystal Form III of the compound of the following chemical formula 1, a pharmaceutically acceptable salt thereof, or a solvate thereof,
In the X-ray powder diffraction (XRPD) pattern, the following diffraction angles (2θ values): 6.238±0.2°, 8.257±0.2°, 8.828±0.2°, 14.637±0 .2°, 16.618±0.2°, 17.465±0.2°, 18.859±0.2°, 19.061±0.2°, 19.333±0.2°, 20 Crystal form III having three or more characteristic peaks selected from .642±0.2°, 22.679±0.2° and 25.985±0.2°.
(In the chemical formula 1,
R ₁ is C ₂ -C ₅ alkyl) Crystal Form III according to claim _{1, wherein said R 1} is C ₂ -C ₄ alkyl. The compound of Formula 1 is N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-( Crystal Form III according to claim 2, which is morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide. Crystalline Form III according to claim 1, wherein the pharmaceutically acceptable salt is selected from the group consisting of hydrochloride, sulfate, nitrate, phosphate, hydrobromide and hydroiodide. . Crystal Form III according to claim 1, which is a crystal form of the formamide solvate of Formula 1. A method for producing crystal form III according to any one of claims 1 to 5, comprising:
Dissolving the compound of Formula 1 in a crystallization solvent to prepare a mixed solution;
and obtaining crystals from the mixed solution. 7. The method for producing crystal form III according to claim 6, wherein the crystals are obtained by an anti-solvent crystallization method. 7. The method for producing crystal form III according to claim 6, wherein the crystals are obtained by a slurry method. 7. The method for producing crystal form III according to claim 6, wherein the crystallization solvent includes a polar organic solvent. 10. The method for producing crystalline form III according to claim 9, wherein the polar organic solvent comprises formamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone, or a mixture thereof. 10. The method for producing crystal form III according to claim 9, wherein the crystallization solvent further includes a nonpolar organic solvent. 12. The method for producing crystalline Form III according to claim 11, wherein the non-polar organic solvent comprises hexane, heptane, cyclohexane, carbon tetrachloride, benzene, chloroform or a mixture thereof. A pharmaceutical composition comprising crystalline Form III according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier. A pharmaceutical composition for enhancing melanocortin-4 receptor function, comprising crystal form III according to any one of claims 1 to 5, and a pharmaceutically acceptable carrier. The pharmaceutical composition according to claim 13, wherein the composition is for the prevention or treatment of obesity, diabetes, inflammation or erectile dysfunction.